Biofilms are surface-attached microbial communities found in clinical, industrial and natural environments. Biofilms negatively impacts human health, in particular the formation of antibiotic resistant biofilms on a broad range of medical implants such as catheters, orthopedic implants and contact lenses is a significant clinical problem. Our previous studies demonstrated that the nucleotide signal cyclic diguanylate (cdG) controls biofilm formation via control of extracellular polysaccharide (EPS) and flagellar function. In this renewal application, we analyze a signaling pathway required for the control of flagellar function during early biofilm formation by the opportunistic pathogen P. aeruginosa. Our central hypothesis is that cdG levels are up-regulated in response to cell-to-substratum contact, leading to reduced motility and promotion of biofilm formation. In particular, we propose a surface-sensing pathway that depends on the PilY1 protein, with subsequent increase of cdG, dynamic swapping of two distinct flagellar stators triggered by increased cdG, and, finally, reduced motility, favoring biofilm formation. In this application, we use community level and single cell analyses to explore the mechanistic underpinnings of the earliest events in biofilm formation. A more complete understanding of early events in biofilm formation by this opportunistic pathogen, and a more general understanding of cdG signaling, will provide insights into how to prevent and treat infections caused by a broad range of biofilm-forming microbes. Our Specific Aims are: Specific Aim 1. Test the hypothesis that early biofilm formation is promoted by surface-dependent stimulation of cdG levels mediated by PilY1, a component of a putative surface-sensing pathway. Specific Aim 2. Test the hypothesis that a newly described signal transduction pathway regulates early biofilm formation through production and binding of cdG to control flagellar-mediated surface motility. Specific Aim 3. Test the hypothesis that dynamic stator exchange down-regulates flagellar motility on a surface in response to cdG.